Studies on the Reaction Mechanism of the Cytochrome oxidase based on the Crystal Structure

在影响细胞色素c氧化酶结晶条件的诸多因素中，稳定溶解酶蛋白的洗涤剂结构是最关键的。我们发现了7种合成的非离子洗涤剂，它们提供可结晶的酶制剂。迄今为止研究最广泛的一种烷基氧乙烯BL8SY(CH_3（CH_2)_<11>O(CH_2CH_2)_<11>OH）晶体，在6A分辨率下衍射x射线，相关系数大于0.5，在7A分辨率下相关系数大于0.8。偶见x射线衍射高于5A。六方晶体的重原子衍生物由Hg、W和Au化合物组成。为了进行分子置换分析，我们制备了不同空间群的二维晶体，得到的电子衍射显示出与Henderson等人之前给出的三维结构有明显不同。此外，二聚体的形成明显影响了单体的三维结构。酵母和细菌（副球菌）酶的制备方法得到了改进，在纯度和可重复性方面比以前发表的方法要好得多。细菌酶产生了二维晶体。我们得到的酵母制剂可溶于任何中性缓冲液，不需要任何洗涤剂。这一特性在三维结晶方面很有前景。基本建立了羰基细胞色素c氧化酶单晶红外各向异性的测定方法。其中最关键的是如何制作出小而光滑的CaF_2管，用于单晶在不同方向上的红外和光学测量。在研究过程中，研究了完全还原和完全还原CO的结晶条件，得到了x射线衍射结果。所得衍生物的空间基团和晶格常数与静止氧化态的空间基团和晶格常数在实验精度范围内一致，说明电子转移和配体结合过程中的构象变化并不大到影响酶分子的四级结构。因此，一旦在一定分辨率下获得氧化态晶体，可能通过用适当的氧化剂、还原剂或配体处理晶体，可以很容易地在其他氧化和结合状态下获得具有相同质量的晶体。少

Among many factors affecting crystallization conditions of cytochrome c oxidase, structure of detergent for stabilizing the solubilized enzyme protein has turned out to be the most critical. We have discovered seven species of synthetic non ionic detergents which provide crystallizable enzyme preparations. An alkyloxyethylene, BL8SY(CH_3(CH_2)_<11>O(CH_2CH_2)_<11>OH), most extensively examined so far, gave tetragonal crystals which diffract x-ray with the correlation coefficient larger than 0.5 at 6A resolution and 0.8 at 7A resolution. Occasionally X-ray diffraction was found at higher than 5A. Heavy atom derivatives of the hexagonal crystal were obtained by Hg, W and Au compounds. For molecular replacement analysis, 2D crystals in various space groups were prepared and the electron diffractions obtained indicates a three dimensional structure significantly different from that previously given by Henderson et al. Furthermore, formation of dimer affects clearly the 3D structure of the … More monomer.Preparation methods of yeast and bacterial(Paracoccas)enzymes have improved to establish much better methods than previously published ones in terms of the yeald, purity and reproducibility. The bacterial enzyme gave two dimensional crystals. The yeast preparation we obtained is soluble to any neutral buffer without any detergent. This property is quite promising for 3D crystallization.Method for infrared anisotropy of the single crystal of carbonyl cytochrome c oxidase has been essentially established. The most critical point was how to make small and polished tubes of CaF_2 for both infrared and optical measurements for a single crystal at various direction. During this investigation, crystallization conditions for fully reduced and fully reduced CO forms were examined to obtain X-ray diffraction. The space groups and lattice constants of these derivatives obtained coincide with those of the resting oxidized form within the experimental accuracy, suggesting that the conformational changes during the electron transfer and ligand binding are not so large as affecting quaternary structure of the enzyme molecule. Thus, once a crystal at an oxidation state are obtained at a certain level of resolution, crystals with the same quality may be easily obtained at other oxidation and binding states probably by treatment of the crystal with appropriate oxidant, reductant or ligand. Less